PROJECT SUMMARY/ABSTRACT Elderly individuals are faced with a significant burden of hematologic diseases, including anemia, clonal hematopoiesis, myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). MDS are a group of blood diseases of the elderly that initiate in a HSC and frequently progress to AML. While mutations in tumor suppressor p53 are found in the blood cells of aged healthy individuals and in MDS patients, the role of mutant p53 in HSC aging and pathogenesis of MDS are largely unknown. We recently discovered that some gain-of- function (GOF) mutant p53 proteins enhanced the self-renewal potential of HSCs by a novel mechanism involving disruption of epigenetic pathways. Our objective here is to characterize the role of GOF mutant p53 in HSC aging and pathogenesis of MDS and identify novel therapeutic targets for the treatment of MDS. We hypothesize that acquisition of specific GOF p53 mutations in aged HSCs drives the development of pre- leukemic hematopoietic stem cells with enhanced self-renewal capability, thereby allowing clonal evolution and subsequent acquisition of mutations and/or epigenetic changes that lead to the formation of MDS stem cells. We speculate that pharmacological inhibition of pathways synthetic lethal to p53 mutations will eliminate drug- resistant MDS stem cells and improve treatment outcome. In this proposed research, we will utilize biochemical, genetic, molecular, and pharmacological approaches as well as vertebrate models of human MDS to investigate the epigenetic regulation of stem cell aging and MDS development. We will employ unbiased genome-wide approaches, including RNA-seq, ChIP-seq, and DNA methylation assays, to identify GOF mutant p53 targets and decipher how GOF mutant p53 regulates gene expression in aged HSCs. In addition, we will characterize MDS stem cells in p53R248W/+Asxl1+/- mice and elucidate the mechanism by which mutant p53 enhances MDS stem cell self-renewal. Further, we will identify genes and pathways that are synthetic lethal to mutant p53 in human MDS cells using CRISPR-Cas9 screening. Our results will fill a significant gap regarding the origin and development of pre-leukemic hematopoietic stem cells and MDS stem cells. We anticipate that these studies will delineate the effects of specific mutant p53 proteins in aged HSCs, pre-leukemic HSCs, and MDS stem cells, and will likely identify novel targets in human MDS cells with p53 mutations that may have therapeutic potential for eliminating drug-resistant MDS stem cells. These outcomes would likely open new avenues for aging, MDS, and cancer research.